Proteins at work – rapid freeze quench

Many proteins undergo large structural changes as they function. X-ray crystallography offers detailed snap-shots of single states, but alternative methods are necessary to capture the range of structures occurring in solution. By affording accurate measurements of the distance between two paramagnetic centers, EPR spectroscopy in combination with suitable tags offers unique opportunities to investigate the structural changes that proteins can undergo. Here we focus on proteins in equilibrium conditions with and without substrate binding that are responsible to the conformational change. In addition we are interested in trapping intermediate conformations that appear during the protein function and are important for elucidating the mechanism of the protein/enzyme.  For this purpose we have developed a microfluidic freeze quench system that is designed for trapping samples for W-band pulse EPR measurements, minimizing the amount of sample needed.

In this context we study RNA helicases that bind and remodel RNA in an ATP-dependent manner. We concentrate on DEAD-box protein A (DbpA) from Escherichia coli and its Bacillus subtilis  homolog, YxiN, which are unique among the DEAD proteins because their ATPase activity is strongly enhanced by the presence of an RNA molecule that contains hairpin 92 of 23S ribosomal RNA. The various steps in the mechanism of DbpA were well characterized in terms of necessary cofactors, kinetic and thermodynamic parameters, but the underlying, structural basis is still  missing.  We are exploring  different conformational states of proteins trapped  by non-hydrolysable ATP analogs and we performing time resolved rapid freeze quench (RFQ) measurements to trap intermediates using ATP.  Other proteins we are investigating are drug transporters,  the mechanism of which involves a sequence of conformational changes.



  • Kaufmann, R., Yadid, I. Goldfarb, D., A novel microfluidic rapid freeze-quench device for trapping reactions intermediates for high field EPR analysis. Journal of Magnetic Resonance 2013. 230, 220-226.
  • Kaminker, I.; Sushenko, A.; Potapov, A.; Daube, S.; Akabayov, B.; Sagi, I.; Goldfarb, D. Probing Conformational Variations at the ATPase Site of the RNA Helicase DbpA by High-Field Electron-Nuclear Double Resonance Spectroscopy. Journal of the American Chemical Society 2011, 133, 15514-15523.